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. Author manuscript; available in PMC: 2010 Oct 15.
Published in final edited form as: Biol Psychiatry. 2009 Jul 3;66(8):785–794. doi: 10.1016/j.biopsych.2009.05.021

Figure 3. Glutamatergic neurons are predominant cell type affected by ethanol withdrawal in the hippocampus.

Figure 3

To investigate which cell type is primarily affected by EW we analyzed the phenotype of TUNEL-positive cells in the CA1 region of wild-type mice two days after withdrawal. To this end we performed co-labeling using antibodies against neuron-specific antigen NeuN and astrocyte-specific antigen glial fibrillary acidic protein (GFAP). Panels A-D show two TUNEL-positive cells with chromatin condensation visualized by the DAPI staining. One of these cells (arrowhead) colocalized with NeuN and therefore was identified as a neuron. The second cell (arrow) was NeuN-negative. Panels E-H show a GFAP-positive, damaged astrocyte (arrow). We found that 74.4% of dying cells were neurons (NeuN-positive) and 14.6% were astrocytes (GFAP-positive; quantified in I). Immunohistochemistry for VGLUT1 (a marker of excitatory neurons) and glutamic acid decarboxylase-67 (GAD67; a marker for inhibitory interneurons) in conjunction with TUNEL staining revealed that the majority of dying cells were VGLUT1-positive and GAD67-negative excitatory neurons (J-O). GAD67-positive cells (bright red in P as opposed to punctuate red GAD67-positive synaptic buttons found on most CA1 pyramidal neurons; see J) were TUNEL-negative (P-S). Quantification of the above results shown in T. *** p<0.001. The above estimation was performed using paraffin sections containing CA1 regions of four mice. The results are presented as mean ± SEM.